Helicases are molecular motors that couple nucleotide binding and hydrolysis to nucleic acid unwinding and translocation. Many organisms encode multiple helicases that are essential to fundamental cellular functions such as DNA replication, repair, recombination, transcription and translation. Several human genetic disorders have also been linked to mutations in DNA helicases and helicases from human pathogen are being actively pursued as drug target. We propose to study the molecular mechanisms of two classes of helicases using the powerful single molecule fluorescence techniques. Our approaches have already yielded several surprises and provided previously unattainable data on complex biochemical processes. For example, using single molecule fluorescence resonance energy transfer (FRET), we discovered that PcrA helicase reels in DNA in single base steps, forming a DNA loop, and at the same time efficiently removing other proteins bound to the DNA (Park et al, Cell, 2010). We also discovered that NS3 helicase from a human pathogen, hepatitis C virus, unwinds DNA using a spring-loaded mechanism (Myong et al, Science, 2007). On superfamily 1 helicases (Rep/UvrD/PcrA), we aim to (1) uncover the mechanism and functional importance of chemo-mechanical coupling between ATP hydrolysis and DNA translocation, (2) correlate DNA unwinding and enzyme conformational changes using a hybrid instrument combining ultrahigh resolution optical tweezers with single fluorophore detection, (3) probe consequences of an encounter between a helicase and other DNA bound proteins via imaging-based localization. On replicative hexameric helicases, we aim to (1) determine the step size of DNA unwinding and inter-subunit coordination of T7 gp4, DnaB-like helicase G40P and SV40 Large T antigen (LTag), (2) investigate the mechanism of helicase slipping and how other interacting proteins modulate the slipping events, (3) test if the ring-shaped helicase rotates around the DNA axis during its translocation along double stranded DNA, and (4) probe for a potential coordination two hexamers of LTag unwinding from the origin sequence in opposite directions using four-color single molecule FRET. In all of our studies, bulk-phase biochemical experiments and structural data through collaboration will help us design and interpret our measurements.